Intravenous ascorbate (ascorbic acid, vitamin C), but not oral ascorbate, produces high plasma concentrations in the range that is cytotoxic to pancreatic cancer cells. Though ascorbate has been utilized in cancer therapy, little is known about the mechanism of action. Recent studies from our group have demonstrated that ascorbate induces cytotoxicity and oxidative stress in pancreatic cancer cells and this cytotoxicity appears to be more prominent in tumor vs. normal cells. Pharmacological ascorbate has been hypothesized to be a pro-drug for formation of hydrogen peroxide (H2O2). H2O2 may directly damage mitochondria so that ATP production decreases. Ascorbate-induced oxidative stress could represent a difference between tumor cell and normal cell metabolism amenable to manipulations designed to improve cancer therapy. To gain a mechanistic understanding of ascorbate-induced oxidative stress in pancreatic cancer cells, we will test the hypothesis that production of H2O2 mediates the increased susceptibility of pancreatic cancer cells to ascorbate-induced metabolic oxidative stress, relative to normal human cells. We will test our hypothesis with the following three Specific Aims: 1. Determine if pharmacological ascorbate-induced cytotoxicity is due to pancreatic cancer cell mitochondria sensitivity to H2O2 resulting in decreased ATP production. 2. Determine if pharmacological ascorbate-induced cytotoxicity can be enhanced by compounds that increase the production of hydroperoxides and/or chemical inhibitors of hydroperoxide detoxification in human pancreatic cancer cells in vitro and in vivo. 3. Determine if pharmacological ascorbate-induced cytotoxicity can be enhanced by chemo- therapeutic agents (gemcitabine, 5-FU) in human pancreatic cancer cells in vitro and in vivo. If we can rigorously demonstrate that pharmacological ascorbate induces preferential cytotoxicity and oxidative stress in human pancreatic cancer cells vs. normal human cell types and that this is dependent on the increased production of H2O2, then the results of this proposed research program will provide a foundation for the rational design of a combined modality cancer therapy based on a fundamental difference between the biochemistry of oxidative metabolism in normal vs. pancreatic cancer cells. PUBLIC HEALTH RELEVANCE: Intravenous ascorbate produces high plasma concentrations in the range that is cytotoxic to pancreatic tumor cells. Pharmacological ascorbate has been hypothesized to be a pro-drug for formation of hydrogen peroxide (H2O2). H2O2 may directly damage mitochondria so that ATP production decreases. Our proposal investigates the mechanism by which ascorbate-induces cytotoxicity in pancreatic cancer cells.